1、Designation:E 2146 01Standard Guide forDetection of Nucleic Acid Sequences of the HumanImmunodeficiency Virus HIV-1 by the Polymerase ChainReaction Technique1This standard is issued under the fixed designation E 2146;the number immediately following the designation indicates the year oforiginal adop
2、tion or,in the case of revision,the year of last revision.A number in parentheses indicates the year of last reapproval.Asuperscript epsilon(e)indicates an editorial change since the last revision or reapproval.INTRODUCTIONThis guide covers the amplification and detection of nucleic acids ribonuclci
3、c acid(RNA)anddeoxyribonucleic acid(DNA)of the human immunodeficiency virus type 1(HIV-1)by thepolymerase chain reaction(PCR)technique.PCR is used in clinical laboratories to aid in the diagnosesof genetic and infectious diseases;it is also used as a general tool in molecular biology andbiotechnolog
4、y laboratories.PCR involves the replication,or amplification,of a fragment of DNA byas many as several million times in only several hours.The amplified DNA fragments,which areidentical or nearly identical,can then be detected,identified and quantified by classical procedures ofmolecular biology and
5、 biochemistry.An advantage of PCR is that as few as several molecules of targetRNA or DNA in a biological test specimen can be rapidly and accurately amplified and subsequentlyidentified.The value of using PCR to detect HIV is the rapidity of the procedure(1 day foramplification and detection).Rapid
6、 clinical intervention can delay disease progression and preventopportunistic infections.Acquired immune deficiency syndrome(AIDS)is the end-stage outcome of infection with HIV.Itis one of the leading causes of human death in the world.As of 1998,about 12 million people havedied fromAIDS.In the Unit
7、ed States,until 1996,AIDS was the leading cause of death among persons25 to 44 years of age(1)2.In 1996 the total number of HIV-infected adults and children worldwidewas over 20 million(1).At present two serotypes of HIV are recognized:HIV-1 and HIV-2.HIV-1 is the primary serotypefound in the United
8、 States;two principal genetic groups have been identified:M(main)and O(outlying).Subtype M is further classified into 10 envelope subtypes,A through J.HIV-2 is primarilyrestricted to West Africa.At present,no generally accepted primers exist for the detection of HIV-2nucleic acids.This guide was dev
9、eloped by Subcommittee E48.02 on Characterization and Identification ofBiological Systems in collaboration with DIN(German Institute for Standardization)Committee E9on Serodiagnosis of Infectious Diseases and Diseases of the Immune System of DINs Department forMedical Standards(Normenausschuss Mediz
10、in).It is recommended that this HIV-specific PCR guidebe used in conjunction with ASTMs general PCR guide E 1873(Standard Guide for Detection ofNucleic Acid Sequences by the Polymerase Chain Reaction Technique).The combination of the twoguides provides guidelines,recommendations,basic considerations
11、,criteria,and principles that shouldbe employed when developing,utilizing or assessing PCR protocols for the detection of the RNA orDNA form of HIV nucleic acid.This guide assumes a basic knowledge of virology and molecular biology.It assumes theavailability of,and the ability to search the literatu
12、re for,HIV target-specific PCR protocols.1.Scope1.1 This guide covers considerations,criteria,principles andrecommendations that should be helpful when developing,1This guide is under the jurisdiction ofASTM Committee E48 on Biotechnologyand is the direct responsibility of Subcommittee E48.02 on Cha
13、racterization andIdentification of Biological Systems.Current edition approved May 10,2001.Published June 2001.1Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959,United States.utilizing,or assessing PCR protocols to amplify and detectDNA(by PCR)or RNA by
14、reverse transcriptase PCR(RT-PCR)from HIV.This guide is not a specific protocol for thedetection of HIV.It is intended to provide information that willassist the user in obtaining quality and reliable data.The guideis closely related to and should be used concurrently with thegeneral PCR guideline E
15、 1873.1.2 This guide has been developed for use in any molecularbiology or biotechnology laboratory.This includes but is notlimited to clinical and diagnostic laboratories involved withHIV detection.1.3 This guide does not cover details of the various methodssuch as gel electrophoresis,that can be u
16、tilized to identifyPCR-amplified HIV nucleic acid sequences,nor does it coverdetails of instrument(thermocycler)calibration.1.4 This guide does not cover specific variations of the basicPCR or RT-PCR technology(e.g.,quantitative PCR,multiplexPCR and in situ PCR).1.5 This guide does not address the additional consider-ations necessary for the performance and validation of aquantitative PCR test for HIV.NOTE1Warning:Laboratory work involving certain clinical speci-mens and microorganisms can be ha
